1. Field of the Invention
This invention relates to the microbial production of 3-hydroxypropionaldehyde from glycerol under aerobic conditions using a strain of Klebsiella pneumoniae.
2. Description of the Art
Approximately 10 percent of all petroleum and natural gas consumed in the United States is used in the production of organic chemicals. Acrylic acid, an industrially important polymerizable monomer useful in the manufacture of synthetic polymers and plastics is currently derived from petroleum. To reduce dependence on fossil oil reserves, new energy-efficient routes are needed that utilize renewable resources as feedstocks for the synthesis of acrylic acid and other petroleum-derived chemicals. Glycerol which is easily accessible from ubiquitous plant and animal lipids, is such a feedstock. While it is known that acrylic acid can be prepared by chemical oxidation of 3-hydroxypropionaldehyde (3-HPA), presently, no commercial source of 3-HPA exists. One suggested potential source for 3-HPA is through fermentation of glycerol. Abeles et al. (Biochim. Biophys. Acta. 41: 530-531 (1960)) positively identified 3-HPA as a metabolic intermediate in the conversion of glycerol to trimethylene glycol by Klebsiella oxytoca NRRL B-199 (previously classified K. pneumoniae and Aerobacter aerogenes) grown anaerobically on glycerol. Normally, 3-HPA is not an end product of bacterial glycerol dissimilation, but an intermediate which must be trapped and so forced to accumulate by manipulation of the metabolism. Abeles et al., supra, noted that 3-HPA accumulated when anaerobic fermentation of glycerol was carried out in the presence of semicarbazide. Fermentation studies carried out by Slininger et al. (Applied and Environmental Microbiology 46(1): 62-67 (1983)) using K. oxytoca NRRL B-199 under aerobic conditions determined that 55% of the theoretical maximum concentration of 3-HPA could be realized by fermentation of glycerol (30 g/liter) when semicarbazide hydrochloride concentration was optimized at 26.8 g/liter. Further studies by Slininger et al. (Applied and Environmental Microbiology, 50(6): 1444-1450 (1985)) indicated that cell growth and glycerol dehydratase induction required at least 48 to 72 hours, and peak 3-HPA accumulation reached 15-20 g/liter at a specific production rate of 0.1 to 0.3 gram/gram biomass/hour.
Pathways responsible for glycerol dissimilation by K. oxytoca have been described. After transport into the cell, glycerol dissimilation is believed to occur via either of two inducible pathways summarized in FIG. 1, the glycerophosphate (glp) or the dihydroxyacetone (dha) system. 3-HPA is an intermediate of the dha path which coproduces trimethylene glycol (TMG) and dihydroxyacetone phosphate via NAD-coupled reactions. Regulatory mechanisms governing the course of dissimilation are complex. Carbon source and hydrogen acceptors such as oxygen are influential. Respiratory repression of enzyme induction may regulate use of the glp versus dha pathway in K. oxytoca strains. Oxygen-mediated deactivation of sYnthesized dehydratases and dehydrogenases may also influence observed enzyme activities.
Deviations from common regulatory mechanisms documented for K. oxytoca strains, such as NRRC B-199 (ATCC 8724), have been reported (Slininger et al, 1985, supra). Attempts to convert glycerol to 3-HPA by glycerol dehydratase isolated from Lactobacillus sp. strain NRRL B-1720 were unsatisfactory because enzyme activity was lost within 60 to 90 minutes after the reaction initiation (Slininger et al., 1983, supra).